HIV encodes a number of accessory genes not commonly found in other retroviruses. The purpose of this project is to investigate the biological and biochemical functions of the HIV accessory proteins Vif and Vpu and to understand their precise role in virus replication. One of our goals is to characterize cellular factors involved in Vif or Vpu function. From our studies on Vpu we expect to gain insights into general principles of protein degradation from the ER and into mechanisms involving late stages of virus production, in particular the involvement of ion channels in the secretory pathway. As a result of our experiments on Vif, we not only expect to gain insights into the function of this viral factor but we expect to learn more about the role of the cytoskeleton in virus replication. Finally, we hope our research will provide a basis for the assessment of viral proteins as potential antiviral targets.The vpu gene is unique to HIV-1 and encodes a small integral membrane protein. Vpu regulates virus release from the cell surface and degradation of CD4 in the endoplasmic reticulum. These two biological activities of Vpu are based on two independent and distinct molecular mechanisms that can be attributed to separable structural domains of Vpu. Vpu-regulated virus release is sensitive to changes in the transmembrane (TM) domain of Vpu and is correlated with an ion channel activity of Vpu. CD4 degradation, on the other hand, involves a direct interaction of the Vpu and CD4 cytoplasmic domains. We demonstrated in the past that Vpu-mediated CD4 degradation involves the ubiquitin-dependent proteasome pathway and requires an interaction with a novel cellular protein, betaTrCP. BetaTrCP was found to engage in ternary complexes with Vpu and CD4 - requiring phosphorylation of Vpu ? and represents a direct link to Skp1p, a known component of E3 ubiquitin ligase complexes. We now have evidence that TrCP localizes both to the cytoplasm and the nucleus of a cell and that the protein is phosphorylated at several positions. Research projects to investigate the function of TrCP phosphorylation are currently underway. Preliminary evidence suggests that phosphorylation of TrCP is more prominent in the cytosolic fraction suggesting a possible role of TrCP phosphorylation in the intracellular distribution of the protein. To further characterize the role of Vpu for viral replication, we have established cell lines with inducible Vpu expression and we are in the process of analyzing the effects of Vpu on cellular processes.HIV-2, which is very closely related to HIV-1, lacks a vpu gene. However, we found that in the absence of the HIV-2 Env glycoprotein, Vpu can enhance HIV-2 virus release. In the presence of HIV-2 Env, efficient virus release is observed which was not further enhanced by Vpu. We subsequently observed that some (but not all) of the HIV-2 Env proteins encode an activity that regulates virus particle release, in a Vpu-like manner. A similar Vpu-like function was observed for at least one macrophage-tropic HIV-1 isolate. Experiments designed to identify domains in HIV-2 Env that are critical for the Vpu-like function ruled out an involvement of the cytoplasmic domain of Env. In contrast, mutation of a single amino acid in the ectodomain of an inactive isolate was sufficient to activate (or restore) its Vpu-like activity. Preliminary evidence suggests that the ability or inability of HIV-2 Env to regulate virus release is associated with its potential to efficiently form homo-oligomers.Vif is a 23-kDa basic protein, which has an important function in regulating infectivity of progeny virions. The biochemical mechanism of Vif function is obscure. We analyzed the role of Vif by studying its subcellular distribution by cell fractionation as well as confocal microscopy. We found that a substantial portion of intracellular Vif protein is associated with the cytoskeleton, specifically intermediate filaments. The association of Vif with intermediate filaments is specific and can result in the reorganization of the cytoskeletal network. In addition, we found that Vif is incorporated into virus particles where it associates with the core of virions. Mutational analysis of Vif suggests a role for a N- terminal domain in Vif for the interaction with vimentin. Preliminary evidence also points to an interaction of Vif with viral or cellular RNA, presumably involving a central domain in Vif. Deletion of this central domain in Vif severely affects its subcellular distribution and inhibits virion incorporation of Vif.